The ultimate goal of a vaccination is to achieve with minimal side effects a long-lasting, fully protective immune response against a pathogen. Techniques developed over the last decade have allowed the construction of novel types of vaccines that no longer rely on the use of inactivated or attenuated infectious agents, but on individual proteins or even fragments thereof. Before a subunit vaccine can be designed, the different types of immune responses to the different proteins of the infectious agent, and their role in conferring protection have to be elucidated. During the last granting period, we concentrated on defining immune responses to the two internal proteins of rabies virus, i.e., the nucleoprotein and the nominal phosphoprotein. In this application, we propose an in-depth study of immune responses to the viral glycoprotein that is apparently the only viral antigen capable of inducing immune effector mechanisms that can limit the spread of rabies virus once it reaches the central nervous system. Furthermore, we intend to test different prototype vaccines, based on the rabies virus glycoprotein, for induction of protective immune responses. The application is divided into three specific aims. In the first aim, T cell epitopes of the glycoprotein will be defined, the influence of glycosylation of synthetic peptides carrying immunodominant T cell epitopes on their ability to stimulate T cells, especially in vivo, will be tested. The influence of physiological glycosylation patterns of the rabies virus glycoprotein in inducing immune responses will be investigated as well. In the second specific aim, the different immune effector mechanisms to the rabies virus glycoprotein, and their role in providing protection against a peripheral and intracerebral challenge, will be tested using knock-out mice that lack the ability to develop defined types of immune responses. in the third specific aim, we will use vectors expressing the rabies virus glycoprotein to transiently express this protein in vivo. The immune responses induced upon vector immunization will be characterized. Attempts will be made to improve the efficacy by using different expression vectors and different routes of immunization. The duration of expression of the viral antigen and the persistence of vector DNA in mammalian cells will be determined. The effect of co-expressing other immunologically relevant proteins such as cytokines will be investigated.